The complex group of opioid receptors are biologically and clinically important in mediating the action of endogenous and exogenous opioids. Despite the clinical advantages of developing drugs which serve as specific agonists and antagonists for opioid receptor subtypes, studies to date have been limited by the inability to satisfactorily isolate and characterize these multiple opioid receptors. This project is aimed at better defining the biochemical and molecular basis of opioid systems and actions. The opioid receptor system is believed to consist of at least an opioid ligand binding receptor protein, and an inhibitory guanine nucleotide regulatory system (Gi) which regulates the receptor affinity and possibly serves as a signal transducer to produce cellular biochemical responses such as inhibition of adenylate cyclase and of neurotransmitter release. Based on this model, we plan to define the role of Gi in the opioid receptor system. We have successfully purified substantial quantities of Gi from rat brains. We propose to further characterize this Gi protein, to reconstitute Gi back to membranes containing "uncoupled" opioid receptors, to use this Gi as a tool to purify solubilized opioid receptors, and to study possible mechanisms of tolerance that involve Gi. We have recently established a rat spinal tolerance model where we can manipulate to achieve selective as well as cross tolerance to specific Mu- and Delta-agonists. The Mu- and Delta-receptor binding activities and opioid sensitive GTPase activity will be examined in these spinal cord membranes to correlate with the development of opioid tolerance. The spinal membranes from tolerant animals will also be used in reconstitution experiments to study the role of G-proteins in opioid tolerance. As shown in the Preliminary Studies we have isolated a large quantity of Gi- and Go-protein from rat brains. We will attempt to further separate Go and Gi and to study their individual roles in the opioid receptor systems. Information generated from these studies will add to the elucidation of the molecular basis of the opioid receptor system and the mechanism of opioid actions.